Original Articles

The nature of ischemia/reperfusion injury

The most common causes of death after heart transplantation (HTx) include acute rejection and multi-organ failure in the early period and malignancy and cardiac allograft vasculopathy (CAV) in the late period. Polyclonal antibody preparations such as rabbit anti-thymocyte globulin (ATG) may reduce early acute rejection and the later occurrence of CAV after HTx. ATG therapy depletes T cells, modulates adhesion and cell-signaling molecules, interferes with dendritic cell function, and induces B-cell apoptosis and regulatory and natural killer T-cell expansion. Evidence from animal studies and from retrospective clinical studies in humans indicates that ATG can be used to delay calcineurin inhibitor (CNI) exposure after HTx, thus benefiting renal function, and to reduce the incidence of CAV and ischemia-reperfusion injury in the transplanted heart. ATG may reduce de novo antibody production after HTx. ATG does not appear to increase cytomegalovirus infection rates with longer prophylaxis (6–12 months). In addition, ATG may reduce the risk of lymphoproliferative disease and does not appear to confer an additive effect on acquiring lymphoma after HTx. Randomized, controlled trials may provide stronger evidence of ATG association with patient survival, graft rejection, renal protection through delayed CNI initiation, as well as other benefits. It can also help establish optimal dosing and patient criteria to maximize treatment benefits.

Several studies have been carried out in the last 30 years in the attempt to clarify the possible role of glutamate as a neurotransmitter/neuromodulator in the gastrointestinal tract. Such effort has provided immunohistochemical, biomolecular and functional data suggesting that the entire glutamatergic neurotransmitter machinery is present in the complex circuitries of the enteric nervous system (ENS), which participates to the local coordination of gastrointestinal functions. Glutamate is also involved in the regulation of the brain-gut axis, a bi-directional connection pathway between the central nervous system (CNS) and the gut. The neurotransmitter contributes to convey information, via afferent fibers, from the gut to the brain, and to send appropriate signals, via efferent fibers, from the brain to control gut secretion and motility. In analogy with the CNS, an increasing number of studies suggest that dysregulation of the enteric glutamatergic neurotransmitter machinery may lead to gastrointestinal dysfunctions. On the whole, this research field has opened the possibility to find new potential targets for development of drugs for the treatment of gastrointestinal diseases. The present review analyzes the more recent literature on enteric glutamatergic neurotransmission both in physiological and pathological conditions, such as gastroesophageal reflux, gastric acid hypersecretory diseases, inflammatory bowel disease, irritable bowel syndrome and intestinal ischemia/reperfusion injury.

Azadirachta indica is widely distributed in Africa, Asia and other tropical parts of the world. A. indica (AI) is traditionally used for the treatment of several conditions including cancer, hypertension, heart diseases and skin disorders. Intestinal ischaemia-reperfusion is a common pathway for many diseases and may lead to multiple organ dysfunction syndrome and death.

In this study, we investigated the ameliorative effects of AI on intestinal ischaemia-reperfusion injury-induced cardiorenal dysfunction.

Sixty rats were divided into 6 groups; each containing 10. Corn oil was orally administered to group A (control) rats for 7 days without intestinal ischaemia-reperfusion injury. Group B underwent intestinal ischaemia-reperfusion injury (IIRI) without any pre-treatment. Groups C, D, E and F were pre-treated orally for 7 days with 100 mg/kg AI (100 and (200 mg/kg) vitamin C (100 and 200 mg/kg) respectively and thereafter underwent IIRI on the 8th day.

The cardiac and renal hydrogen peroxide increased significantly whereas serum xanthine oxidase and myeloperoxidase levels were significantly elevated (p < 0.05) in IIRI only when compared to the control. The cardiac and renal reduced glutathione, glutathione peroxidase, protein thiol, non-protein thiol and serum nitric oxide (NO) decreased (p < 0.05) significantly following IIRI. Immunohistochemical evaluation of cardiac and renal tissues showed reduced expressions of the extracellular signal regulated kinase (ERK1/2) in rats with IIRI only. However, pre-treatment with A. indica and vitamin C significantly reduced markers of oxidative stress and inflammation together with improvement in antioxidant status. Also, reduced serum NO level was normalised in rats pre-treated with A. indica and vitamin C with concomitant higher expressions of cardiac and renal ERK1/2.

Together, A. indica and vitamin C prevented IRI-induced cardiorenal dysfunction via reduction in oxidative stress, improvement in antioxidant defence system and increase in the ERK1/2 expressions. Therefore, A. indica can be a useful chemopreventive agent in the prevention and treatment of conditions associated with intestinal ischaemia-reperfusion injury.

Protection of intestinal graft mucosa during cold preservation is still an unmet need in clinical practice, thus affecting the success of transplantation. The present study investigates the ability of two ischemic preconditioning (IPC) procedures to limit cold preservation injury. Three groups of Sprague–Dawley rats were recruited (n = 11 each) as follows: the short IPC (SIPC) performed through 4 cycles of mesenteric ischemia of 4 min each followed by 10 min of reperfusion, the long IPC (LIPC) obtained by 2 ischemic cycles of 12 min each followed by 10 min of reperfusion, and the control group (C) without IPC. Grafts were then stored in cold histidine–tryptophan–ketoglutarate solution and samples were taken at 0, 3, 6 and 9 h lasting preservation. Both IPC groups showed an advanced degree of preservation with delayed development of graft mucosa damage, mainly in the crypt region. At the beginning of preservation, the graft mucosa in both IPC groups showed lower degree of mucosal injury index (MII) by 50% in comparison with C group. Specifically, a significant improvement of MII was observed after 3 h of preservation in the LIPC group (p < 0.05) in comparison with untreated C grafts. Significant atrophy of the intestinal mucosa in C group was found after 3 h of preservation (p < 0.01), in SIPC group the progress of atrophy was delayed to 6 h (p < 0.001), and in LIPC group only moderate decrease in that was found. A parallel increase of laminin expression with the MII rate after 6 and 9 h of preservation in comparison with the level at time 0 was observed in all grafts (p < 0.001 and p < 0.01, respectively). In both IPC groups the apoptotic cell (AC) rate was significantly reduced at the beginning of cold preservation (p < 0.05 both). Moreover, in both the SIPC and C groups, the progressive increase in MII rate connected with AC rate decrease was due to a predominance of necrosis. By contrast in the LIPC group, after an increase of nearly 50% in the AC rate at the 3rd hour, its level remained fairly constant during the further 6 h of preservation, thus probably preventing necrosis and improving graft viability.

Minimizing the inflammatory events that follow intestinal transplantation may influence immediate graft function and improve outcome. Ischemic preconditioning (IPc) has been shown to ameliorate early inflammatory responses, and it may also attenuate the potentially damaging inflammation after intestinal transplantation. Herein, we examine the influence of intestinal IPc on inflammatory indices (tissue expression of ICAM-1, CD11a, and CD44 and serum levels of the soluble ICAM-1, sICAM-1) after heterotopic intestinal transplantation.

Lewis rats received full-length preconditioned or non-preconditioned Brown Norway intestinal allografts in the absence of immunosuppression. Preconditioned grafts were subjected to 1 cycle of 10 minutes of ischemia-reperfusion. Preconditioned and non-preconditioned isografts acted as controls. Blood was collected on alternate days post-transplant, and graft tissue harvested on sacrifice. ICAM-1, CD44, and CD11a expression was determined by immunohistochemistry, and the area of staining was quantified using image analysis. Serum soluble ICAM-1 levels were determined using an R&D Systems Quantikine enzyme immunoassay.

(1) IPc ameliorated serum levels of sICAM-1 until severe rejection (day 7) overcame this down-regulation when compared to non-preconditioned allografts (day 3: 34,304 vs 40,479 pg/mL; day 5: 52,441 vs 61,593 pg/mL; day 7: 75,114 vs 73,309 pg/mL; day 9: 72,872 vs 76,314 pg/mL, respectively). (2) ICAM-1 expression was significantly lower in preconditioned allografts (1.02 vs 2.01 mm²). (3) CD44 tissue levels were also found to be lower in preconditioned allografts (0.86 vs 1.13 mm²). (4) There was a significant relationship between tissue ICAM-1 expression and serum levels of soluble ICAM-1 (P < .02).

IPc improves inflammatory indices in the early stages following intestinal transplantation, and this might lead to a preserved cellular, architectural, and functional graft status. Furthermore, our results support the use of soluble ICAM-1 as a marker of endothelial activation, and thence of inflammation and developing rejection.

Rabbit anti-thymocyte globulin (RATG) has been used as induction therapy in heart transplantation. RATG is polyclonal and has been postulated to have anti-humoral properties by preventing the production of circulating antibodies after heart transplant. Thus, we reviewed our patients who received RATG induction therapy and compared them with those who did not receive therapy for post-transplant de novo antibody production.

Between January 1, 2006, and January 1, 2013, we assessed 196 non-sensitized heart transplant recipients and divided them into those who received 3 to 5 days of RATG induction therapy mostly due to renal insufficiency (n = 35) versus patients who did not receive therapy (n = 161). All patients were given tacrolimus, mycophenolate mofetil, and corticosteroids. Post-transplant circulating antibodies were routinely monitored at 1, 3, 6, and 12 months after heart transplantation; 1-year and 3-year end points were assessed.

The RATG-treated group had a significantly higher 12-month freedom from de novo antibody production compared with the patients who did not receive RATG induction (89% vs 71%, log-rank P = .043); however there was no significant difference for 12-month freedom from de novo donor-specific antibody production (91% vs 88%, log-rank P = .541). Treated rejection rates in the first-year were comparable in both groups; 3-year actuarial survival, freedom from cardiac allograft vasculopathy, and freedom from non-fatal major adverse cardiac events were also similar between both groups.

RATG induction therapy appears to reduce the production of de novo circulating antibodies in non-sensitized patients during the first year after heart transplantation. Although there were no short-term clinical differences between groups, there were imbalances in group characteristics and relatively short follow-up, which are limitations to this study. A randomized, clinical trial with longer follow-up in a larger cohort of patients is warranted.